LOCUS       sequence-155821-        7637 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9C-VPR in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7637)
  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 7637)
  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-CMV-Cas9C-VPR
FEATURES             Location/Qualifiers
     source          1..7637
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     promoter        311..514
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     465..485
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          606..738
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      768..777
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      772..781
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1378..1389
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2907..2927
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    2964..2988
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3018..3167
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             3192..3212
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3642..3998
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             4017..4586
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     repeat_region   4686..4815
                     /label=AAV2 ITR
     primer_bind     complement(4845..4862)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4845..4861)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4854..4876)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5003..5458
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5090..5109)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5300..5321
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5485..5589
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5590..5658,5659..6450)
                     /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"
     primer_bind     complement(5808..5827)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6621..7209
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7110..7129
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    7497..7518
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7533..7538,7539..7556,7557..7563)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7571..7587
                     /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     7576..7598
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7595..7611
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7595..7611
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacccg ctagcaggca
      181 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      241 ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      301 tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc ggtttgactc
      361 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      421 tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag
      481 gcgtgtacgg tgggaggtct atataagcag aggtcgttta gtgaaccgtc agatcactag
      541 tagctttatt gcggtagttt atcacagtta aattgctaac gcagtcagtg ctcgactgat
      601 cacaggtaag tatcaaggtt acaagacagg tttaaggagg ccaatagaaa ctgggcttgt
      661 cgagacagag aagattcttg cgtttctgat aggcacctat tggtcttact gacatccact
      721 ttgcctttct ctccacaggg gtaccgaagc cgctagcgct accggtcgcc accatggcgg
      781 cggcgtgccc ggaactgcgt cagctggcgc agagcgatgt gtattgggat ccgattgtga
      841 gcattgaacc ggatggcgtg gaagaagtgt ttgatctgac cgtgccgggc ccgcataact
      901 ttgtggcgaa cgatattatt gcgcataact ctggccaggg ggacagtctt cacgagcaca
      961 tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc gttaaggtcg
     1021 tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt atcgagatgg
     1081 cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg atgaagagga
     1141 ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca gttgaaaaca
     1201 cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg gacatgtacg
     1261 tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat atcgtgcccc
     1321 agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc gataaaaata
     1381 gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa aattattggc
     1441 ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg actaaggctg
     1501 aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag cttgttgaga
     1561 cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac accaagtacg
     1621 atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct aagctggtct
     1681 cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat taccaccatg
     1741 cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa tatcccaagc
     1801 ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa atgatcgcaa
     1861 agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc aatattatga
     1921 attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga ccacttatcg
     1981 aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc
     2041 ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta cagaccggag
     2101 gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa
     2161 aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct tacagtgtac
     2221 tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc
     2281 tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac tttctcgagg
     2341 cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag tactctctct
     2401 ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta
     2461 acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc cactatgaaa
     2521 agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa caacacaaac
     2581 actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg atcctcgccg
     2641 acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag cccatcaggg
     2701 agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg cctgcagcct
     2761 tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag gaggtcctgg
     2821 acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc gacctctctc
     2881 agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtgtcg ccagggatcc
     2941 gtcgacttga cgcgttgata tcaacaagtt tgtacaaaaa agcaggctac aaagaggcca
     3001 gcggttccgg acgggctgac gcattggacg attttgatct ggatatgctg ggaagtgacg
     3061 ccctcgatga ttttgacctt gacatgcttg gttcggatgc ccttgatgac tttgacctcg
     3121 acatgctcgg cagtgacgcc cttgatgatt tcgacctgga catgctgatt aactctagaa
     3181 gttccggatc tccgaaaaag aaacgcaaag ttggtagcca gtacctgccc gacaccgacg
     3241 accggcaccg gatcgaggaa aagcggaagc ggacctacga gacattcaag agcatcatga
     3301 agaagtcccc cttcagcggc cccaccgacc ctagacctcc acctagaaga atcgccgtgc
     3361 ccagcagatc cagcgccagc gtgccaaaac ctgcccccca gccttacccc ttcaccagca
     3421 gcctgagcac catcaactac gacgagttcc ctaccatggt gttccccagc ggccagatct
     3481 ctcaggcctc tgctctggct ccagcccctc ctcaggtgct gcctcaggct cctgctcctg
     3541 caccagctcc agccatggtg tctgcactgg ctcaggcacc agcacccgtg cctgtgctgg
     3601 ctcctggacc tccacaggct gtggctccac cagcccctaa acctacacag gccggcgagg
     3661 gcacactgtc tgaagctctg ctgcagctgc agttcgacga cgaggatctg ggagccctgc
     3721 tgggaaacag caccgatcct gccgtgttca ccgacctggc cagcgtggac aacagcgagt
     3781 tccagcagct gctgaaccag ggcatccctg tggcccctca caccaccgag cccatgctga
     3841 tggaataccc cgaggccatc acccggctcg tgacaggcgc tcagaggcct cctgatccag
     3901 ctcctgcccc tctgggagca ccaggcctgc ctaatggact gctgtctggc gacgaggact
     3961 tcagctctat cgccgatatg gatttctcag ccttgctggg ctctggcagc ggcagccggg
     4021 attccaggga agggatgttt ttgccgaagc ctgaggccgg ctccgctatt agtgacgtgt
     4081 ttgagggccg cgaggtgtgc cagccaaaac gaatccggcc atttcatcct ccaggaagtc
     4141 catgggccaa ccgcccactc cccgccagcc tcgcaccaac accaaccggt ccagtacatg
     4201 agccagtcgg gtcactgacc ccggcaccag tccctcagcc actggatcca gcgcccgcag
     4261 tgactcccga ggccagtcac ctgttggagg atcccgatga agagacgagc caggctgtca
     4321 aagcccttcg ggagatggcc gatactgtga ttccccagaa ggaagaggct gcaatctgtg
     4381 gccaaatgga cctttcccat ccgcccccaa ggggccatct ggatgagctg acaaccacac
     4441 ttgagtccat gaccgaggat ctgaacctgg actcacccct gaccccggaa ttgaacgaga
     4501 ttctggatac cttcctgaac gacgagtgcc tcttgcatgc catgcatatc agcacaggac
     4561 tgtccatctt cgacacatct ctgttttgac aataaaatat ctttattttc attacatctg
     4621 tgtgttggtt ttttgtgtta gctagcgcgt agataagtag catggcgggt taatcattaa
     4681 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     4741 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     4801 cgagcgagcg cgcagcctaa ttaaggcctt aattaaccta attcactggc cgtcgtttta
     4861 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     4921 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     4981 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     5041 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     5101 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     5161 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     5221 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5281 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     5341 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     5401 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tataatttca
     5461 ggtggcatct ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5521 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5581 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5641 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5701 ttgggtgcac gagtgggtta catcgaactg gatctcaata gtggtaagat ccttgagagt
     5761 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5821 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5881 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5941 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     6001 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     6061 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     6121 accacgatgc ctgtagtaat ggtaacaacg ttgcgcaaac tattaactgg cgaactactt
     6181 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6241 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6301 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6361 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6421 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6481 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6541 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6601 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6661 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6721 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     6781 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6841 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6901 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6961 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     7021 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     7081 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     7141 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     7201 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctgcggtttt
     7261 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     7321 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     7381 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     7441 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     7501 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     7561 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     7621 gccagattta attaagg
//
