LOCUS       sequence-194494-        8031 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes dCas9(N) fused to AVPR2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8031)
  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 8031)
  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: pAVPR2_TCS(Q'L)_HA-dCas9(N)_P2A-Puro-WPRE
FEATURES             Location/Qualifiers
     source          1..8031
                     /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             2144..2170
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(2186..2209,2210..2212,2213..3787)
                     /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             3818..3874
                     /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(3881..3900)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4377..4397
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4514..4534)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4932..4943)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5074..5091)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5080..5287
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5296..5436
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5296..5425
                     /label=AAV2 ITR
     rep_origin      5511..5966
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5598..5617)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5808..5829
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5983..6002)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6102..6124
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6162..6180)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6248..6352
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6353..6421,6422..7213)
                     /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(6571..6590)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7384..7972
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7873..7892
                     /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 cgatatgctc atggcctcta ccacaagcgc tgtgcccgga catccatctc ttccaagtct
      961 tccctccaac agctcacagg agaggcctct ggatacccgg gacccacttc ttgcaagagc
     1021 ggagctcgcc ttgctctcca tcgttttcgt tgccgtcgcg ttgagcaatg gtctggtgct
     1081 ggccgccctg gcgcgacgag gccgccgagg ccactgggcg cctattcatg tctttatcgg
     1141 ccatctctgt cttgcagacc tggccgtggc gctgtttcag gtgctccctc aactggcttg
     1201 gaaagccacc gatagattcc gggggccaga cgcactgtgt cgggccgtga aatacctgca
     1261 gatggtggga atgtatgcca gttcatatat gatacttgct atgaccttgg acagacacag
     1321 ggctatctgt cgccccatgc tggcgtatag gcacggttcc ggagcccatt ggaacaggcc
     1381 agttctggtt gcgtgggctt tttccctgct tctgagcttg ccccagttgt ttattttcgc
     1441 acagagaaat gtggagggcg gatctggcgt gaccgattgc tgggcttgct tcgctgagcc
     1501 atggggcaga aggacctacg tgacgtggat tgctctcatg gtgttcgttg ctcccaccct
     1561 ggggatcgcc gcatgccagg tcctgatatt cagagaaata cacgcctcct tggttcctgg
     1621 gccaagcgag cgccctggtg gaagaagacg gggtcgaagg actggctctc caggcgaagg
     1681 cgcccacgta tcagctgcgg tagctaagac cgtacgcatg acactggtca tcgtcgtcgt
     1741 ttacgttctt tgctgggccc cgtttttttt ggtgcagctc tgggcggcct gggaccccga
     1801 agcacccctg gaaggtgctc cattcgttct gctcatgctt ctcgcttcac tgaatagctg
     1861 taccaatcct tggatttacg cctctttctc ctccagtgtt agctccgagc tgcggagtct
     1921 tctctgttgc gcccggggca ggaccccccc aagcctgggt ccacaggacg aatcctgcac
     1981 taccgcctcc agttccctgg ccaaagacac ctcttctatc gataccggtg gacgcacccc
     2041 acccagcctg ggtccccaag atgagtcctg caccaccgcc agctcctccc tggccaagga
     2101 cacttcatcg accggtgaga acctgtactt ccagctaact agttacccat acgacgtacc
     2161 agattacgct ggaggttctg gatccgacaa gaagtacagc atcggcctgg ccatcggcac
     2221 caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa
     2281 ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt
     2341 cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac
     2401 cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt
     2461 ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca
     2521 cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc
     2581 caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct
     2641 gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga
     2701 cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa
     2761 ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc
     2821 tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa
     2881 gaagaatggc ctgttcggca acctgattgc cctgagcctg ggcctgaccc ccaacttcaa
     2941 gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga
     3001 cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc
     3061 caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac
     3121 caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac
     3181 cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga
     3241 ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta
     3301 caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct
     3361 gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca
     3421 gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct
     3481 gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg
     3541 ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat
     3601 caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga
     3661 gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct
     3721 gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg
     3781 aatgagaggt ggcggtggct cgggtaccgg atctggagcc accaattttt ctctgctgaa
     3841 gcaggctggt gatgttgaag aaaaccctgg acctgaattc atgaccgagt acaagcccac
     3901 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt
     3961 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
     4021 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     4081 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     4141 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     4201 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     4261 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     4321 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa
     4381 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     4441 accgcgcacc tggtgcatga cccgcaaggg gtaactaagt aaggatcgat ccaagatatc
     4501 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     4561 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     4621 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     4681 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     4741 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     4801 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaagctga
     4861 cgtcctttcc atggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     4921 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     4981 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     5041 cctccccgcc aattcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
     5101 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     5161 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     5221 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg
     5281 ctggggagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     5341 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     5401 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
     5461 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     5521 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     5581 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     5641 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     5701 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     5761 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5821 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
     5881 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     5941 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     6001 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     6061 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     6121 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     6181 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     6241 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     6301 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     6361 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     6421 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     6481 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     6541 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     6601 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     6661 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     6721 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     6781 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6841 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6901 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6961 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     7021 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat
     7081 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     7141 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     7201 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     7261 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     7321 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     7381 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     7441 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     7501 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     7561 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     7621 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     7681 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     7741 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     7801 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     7861 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     7921 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     7981 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca t
//
