LOCUS       Exported                8078 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Encodes dCas9(N) fused to BDKRB2.
ACCESSION   .
VERSION     .
KEYWORDS    pBDKRB2_TCS(Q'L)_HA-dCas9(N)_P2A-Puro-WPRE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8078)
  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 8078)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8078
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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
     CDS             2191..2217
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2233..3834
                     /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             3865..3921
                     /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(3928..3947)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4424..4444
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4561..4581)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4979..4990)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5121..5138)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5127..5334
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5343..5483
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5343..5472
                     /label=AAV2 ITR
     rep_origin      5558..6013
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5645..5664)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5855..5876
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6030..6049)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6149..6171
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6209..6227)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6295..6399
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6400..7260
                     /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(6618..6637)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7431..8019
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7920..7939
                     /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 ccgccccggc tctgactgac cgcgttactc ccacaggtga
      661 gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta
      721 agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca
      781 ctttttttca ggttggaccg gtgcgccacc atgaagacga tcatcgccct gagctacatc
      841 ttctgcctgg tattcgccga ctacaaggac gatgatgacg ccagcatcga tatgttctca
      901 ccctggaaga tttctatgtt cctttctgtg cgcgaggaca gcgtgcccac aactgcgagt
      961 tttagcgccg acatgcttaa cgtgaccctg cagggcccca ctctcaacgg gacatttgct
     1021 cagtctaaat gtcctcaagt ggaatggctc ggctggttga acactatcca gccccctttc
     1081 ctttgggttc tttttgttct ggccacgctg gaaaacatct ttgttctctc tgtgttttgc
     1141 ctccataaga gctcctgtac ggtggccgaa atttacctgg gtaatttggc cgcagcggat
     1201 ctcattttgg cctgtggcct gcctttctgg gctattacta tcagcaataa ttttgattgg
     1261 ttgtttggcg agaccttgtg tcgcgtggtg aatgcaatta tcagtatgaa tctgtacagc
     1321 tcaatctgct tccttatgct cgtcagcatt gacaggtacc tcgcccttgt aaagactatg
     1381 agcatgggcc gcatgagggg cgtacgctgg gctaagctct atagcctggt gatttggggt
     1441 tgcaccctcc tcctcagttc acctatgctg gtcttccgaa cgatgaagga gtactctgac
     1501 gagggccaca atgttaccgc atgcgttatc agttacccat ctttgatttg ggaggtcttc
     1561 acaaacatgc tgctgaacgt ggtgggtttt ttgctccccc tttctgtgat taccttctgt
     1621 acaatgcaga tcatgcaagt tttgcgaaac aacgaaatgc aaaaatttaa ggagatacag
     1681 accgagcgaa gggccacagt gctggtcttg gtcgttttgc tgttgttcat tatctgttgg
     1741 ctgcctttcc agatcagcac tttcctggac acgttgcaca gactcggaat cctgagtagt
     1801 tgtcaggacg aaaggatcat cgacgttatc actcaaatcg cgtccttcat ggcatactcc
     1861 aatagctgct tgaacccatt ggtgtatgtt attgtgggaa aacggtttag gaagaagagc
     1921 tgggaagtgt accaaggggt gtgccaaaag ggaggctgcc gatcagagcc aatccaaatg
     1981 gaaaatagca tgggtaccct gagaacctcc attagcgtgg agcgacagat ccacaagctc
     2041 caggactggg ctggctctag acagatcgat accggtggac gcaccccacc cagcctgggt
     2101 ccccaagatg agtcctgcac caccgccagc tcctccctgg ccaaggacac ttcatcgacc
     2161 ggtgagaacc tgtacttcca gctaactagt tacccatacg acgtaccaga ttacgctgga
     2221 ggttctggat ccgacaagaa gtacagcatc ggcctggcca tcggcaccaa ctctgtgggc
     2281 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
     2341 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa
     2401 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac
     2461 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
     2521 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc
     2581 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac
     2641 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc
     2701 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac
     2761 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
     2821 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc
     2881 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg
     2941 ttcggcaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac
     3001 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac
     3061 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc
     3121 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg
     3181 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct
     3241 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     3301 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag
     3361 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac
     3421 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga
     3481 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg
     3541 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg
     3601 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac
     3661 ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac
     3721 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     3781 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaggtggc
     3841 ggtggctcgg gtaccggatc tggagccacc aatttttctc tgctgaagca ggctggtgat
     3901 gttgaagaaa accctggacc tgaattcatg accgagtaca agcccacggt gcgcctcgcc
     3961 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     4021 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     4081 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     4141 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     4201 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     4261 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     4321 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     4381 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     4441 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     4501 tgcatgaccc gcaaggggta actaagtaag gatcgatcca agatatcgta ttcttaacta
     4561 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     4621 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     4681 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     4741 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     4801 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     4861 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt cctttccatg
     4921 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     4981 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     5041 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgccaat
     5101 tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     5161 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     5221 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     5281 ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc
     5341 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     5401 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     5461 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     5521 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     5581 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     5641 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     5701 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     5761 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     5821 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     5881 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     5941 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     6001 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     6061 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     6121 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     6181 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     6241 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     6301 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     6361 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     6421 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     6481 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     6541 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6601 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     6661 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6721 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6781 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6841 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6901 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     6961 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     7021 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     7081 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg
     7141 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     7201 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     7261 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     7321 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     7381 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     7441 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     7501 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     7561 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     7621 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     7681 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     7741 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7801 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7861 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7921 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7981 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     8041 ttacggttcc tggccttttg ctggcctttt gctcacat
//
