LOCUS       sequence-158688-        7466 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9N in mammalian cells; expresses gRNA P2 for Pd-l1 
            binding.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7466)
  AUTHORS   Chew WL, Tabebordbar M, Cheng JK, Mali P, Wu EY, Ng AH, Zhu K, 
            Wagers AJ, Church GM
  TITLE     A multifunctional AAV-CRISPR-Cas9 and its host response.
  JOURNAL   Nat Methods. 2016 Sep 5. doi: 10.1038/nmeth.3993.
  PUBMED    27595405
REFERENCE   2  (bases 1 to 7466)
  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"; journalName: "Nat 
            Methods. 2016 Sep 5. doi: 10.1038/nmeth.3993."
COMMENT     Sequence Label: pAAV-SMVP-Cas9N-U6-gRNA P2
FEATURES             Location/Qualifiers
     source          1..7466
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(182..201)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      995..1583
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1484..1503
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1871..1892
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1907..1912,1913..1930,1931..1937)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1945..1961
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     1950..1972
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1969..1985
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1969..1985
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   2012..2141
                     /label=AAV2 ITR
     primer_bind     complement(2195..2215)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     enhancer        2445..2824
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2825..3028
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2979..2999
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          3120..3252
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      3282..3291
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3294..3314
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3339..5054
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     primer_bind     complement(5794..5813)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    5823..5904
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     5848..5867
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        6135..6383
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6135..6155
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6306..6325
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6399..6474
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     repeat_region   6537..6666
                     /label=AAV2 ITR
     primer_bind     complement(6685..6702)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6685..6701)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6694..6716)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6843..7298
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6930..6949)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7140..7161
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7325..7429
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7430..7466,1..32,33..824)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVVMVTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
       61 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aatagtggta
      121 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
      181 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
      241 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
      301 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
      361 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
      421 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
      481 acgacgagcg tgacaccacg atgcctgtag taatggtaac aacgttgcgc aaactattaa
      541 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
      601 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
      661 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
      721 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
      781 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
      841 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
      901 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
      961 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     1021 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     1081 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     1141 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     1201 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     1261 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     1321 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     1381 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     1441 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     1501 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     1561 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     1621 tttgctgcgg ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     1681 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     1741 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     1801 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     1861 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     1921 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     1981 atgaccatga ttacgccaga tttaattaag gctgcgcgct cgctcgctca ctgaggccgc
     2041 ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc
     2101 gcgcagagag ggagtggcca actccatcac taggggttcc ttgtagttaa tgattaaccc
     2161 gccatgctac ttatctacct gcggccgcca ttatggaatg tgtgtcagtt agggtgtgga
     2221 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2281 accagtcccg gtctcctccc atgcatgtca atattggcca ttagccatat tattcattgg
     2341 ttatatagca taaatcaata ttggctattg gccattgcat acgttgtatc tatatcataa
     2401 tatgtacatt tatattggct catgtccaat atgaccgcca tgttggcatt gattattgac
     2461 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     2521 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     2581 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     2641 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     2701 aagtccgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     2761 catgacctta cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     2821 catggtgatg cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg
     2881 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     2941 ggactttcca aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt
     3001 acggtgggag gtctatataa gcagaggtcg tttagtgaac cgtcagatca ctagtagctt
     3061 tattgcggta gtttatcaca gttaaattgc taacgcagtc agtgctcgac tgatcacagg
     3121 taagtatcaa ggttacaaga caggtttaag gaggccaata gaaactgggc ttgtcgagac
     3181 agagaagatt cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct
     3241 ttctctccac aggggtaccg aagccgctag cgctaccggt cgccaccatg gccccaaaga
     3301 agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac tccattgggc
     3361 tcgatatcgg cacaaacagc gtcggctggg ccgtcattac ggacgagtac aaggtgccga
     3421 gcaaaaaatt caaagttctg ggcaataccg atcgccacag cataaagaag aacctcattg
     3481 gcgccctcct gttcgactcc ggggaaacgg ccgaagccac gcggctcaaa agaacagcac
     3541 ggcgcagata tacccgcaga aagaatcgga tctgctacct gcaggagatc tttagtaatg
     3601 agatggctaa ggtggatgac tctttcttcc ataggctgga ggagtccttt ttggtggagg
     3661 aggataaaaa gcacgagcgc cacccaatct ttggcaatat cgtggacgag gtggcgtacc
     3721 atgaaaagta cccaaccata tatcatctga ggaagaagct tgtagacagt actgataagg
     3781 ctgacttgcg gttgatctat ctcgcgctgg cgcatatgat caaatttcgg ggacacttcc
     3841 tcatcgaggg ggacctgaac ccagacaaca gcgatgtcga caaactcttt atccaactgg
     3901 ttcagactta caatcagctt ttcgaagaga acccgatcaa cgcatccgga gttgacgcca
     3961 aagcaatcct gagcgctagg ctgtccaaat cccggcggct cgaaaacctc atcgcacagc
     4021 tccctgggga gaagaagaac ggcctgtttg gtaatcttat cgccctgtca ctcgggctga
     4081 cccccaactt taaatctaac ttcgacctgg ccgaagatgc caagcttcaa ctgagcaaag
     4141 acacctacga tgatgatctc gacaatctgc tggcccagat cggcgaccag tacgcagacc
     4201 tttttttggc ggcaaagaac ctgtcagacg ccattctgct gagtgatatt ctgcgagtga
     4261 acacggagat caccaaagct ccgctgagcg ctagtatgat caagcgctat gatgagcacc
     4321 accaagactt gactttgctg aaggcccttg tcagacagca actgcctgag aagtacaagg
     4381 aaattttctt cgatcagtct aaaaatggct acgccggata cattgacggc ggagcaagcc
     4441 aggaggaatt ttacaaattt attaagccca tcttggaaaa aatggacggc accgaggagc
     4501 tgctggtaaa gcttaacaga gaagatctgt tgcgcaaaca gcgcactttc gacaatggaa
     4561 gcatccccca ccagattcac ctgggcgaac tgcacgctat cctcaggcgg caagaggatt
     4621 tctacccctt tttgaaagat aacagggaaa agattgagaa aatcctcaca tttcggatac
     4681 cctactatgt aggccccctc gcccggggaa attccagatt cgcgtggatg actcgcaaat
     4741 cagaagagac catcactccc tggaacttcg aggaagtcgt ggataagggg gcctctgccc
     4801 agtccttcat cgaaaggatg actaactttg ataaaaatct gcctaacgaa aaggtgcttc
     4861 ctaaacactc tctgctgtac gagtacttca cagtttataa cgagctcacc aaggtcaaat
     4921 acgtcacaga agggatgaga aagccagcat tcctgtctgg agagcagaag aaagctatcg
     4981 tggacctcct cttcaagacg aaccggaaag ttaccgtgaa acagctcaaa gaagactatt
     5041 tcaaaaagat tgaatgtttc gactctgttg aaatcagcgg agtggaggat cgcttcaacg
     5101 catccctggg aacgtatcac gatctcctga aaatcattaa agacaaggac ttcctggaca
     5161 atgaggagaa cgaggacatt cttgaggaca ttgtcctcac ccttacgttg tttgaagata
     5221 gggagatgat tgaagaacgc ttgaaaactt acgctcatct cttcgacgac aaagtcatga
     5281 aacagctcaa gaggcgccga tatacaggat gggggcggct gtcaagaaaa ctgatcaatg
     5341 ggatccgaga caagcagagt ggaaagacaa tcctggattt tcttaagtcc gatggatttg
     5401 ccaaccggaa cttcatgcag ttgatccatg atgactctct cacctttaag gaggacatcc
     5461 agaaagcaca agtttgtctg gctggcgata ctctcattac cctggccgat ggacgacgag
     5521 tgcctattag agaactggtg tcacagcaga atttttccgt gtgggctctg aatcctcaga
     5581 cttaccgcct ggagagggct agagtgagta gagctttctg taccggcatc aaacctgtgt
     5641 accgcctcac cactagactg gggagatcca ttagggccac tgccaaccac cgatttctca
     5701 cacctcaggg ctggaaacga gtcgatgaac tccagcctgg agattacctg gctctgccta
     5761 ggagaatccc tactgcctcc tgatctcgag ctcgatgagt ttggacaaac cacaactaga
     5821 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     5881 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     5941 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtacttaa
     6001 gagggggaga ccaaagggcg agacgttaag gcctcacgtg acatgtgagc aaaaggccag
     6061 caaaatgtac aaaaaagcag gctttaaagg aaccaattca gtcgactgga tccggtacca
     6121 aggtcgggca ggaagagggc ctatttccca tgattccttc atatttgcat atacgataca
     6181 aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa
     6241 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     6301 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     6361 tcttgtggaa aggacgaaac accgctaaag tcatccgcgt tttagagcta gaaatagcaa
     6421 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
     6481 ttctagaggg cggccgcagg tagataagta gcatggcggg ttaatcatta actacaagga
     6541 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     6601 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     6661 gcgcagcctt aattaaccta attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     6721 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     6781 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     6841 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     6901 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     6961 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     7021 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     7081 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     7141 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     7201 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     7261 taacgcgaat tttaacaaaa tattaacgtt tataatttca ggtggcatct ttcggggaaa
     7321 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     7381 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     7441 acatttccgt gtcgccctta ttccct
//
