LOCUS       sequence-208555-        6609 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV plasmid expressing CjCas9 under control of CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6609)
  TITLE     Alex Hewitt Lab plasmids
REFERENCE   2  (bases 1 to 6609)
  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"
COMMENT     Sequence Label: pX551-CMV-CjCas9
FEATURES             Location/Qualifiers
     source          1..6609
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        147..526
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        527..730
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     681..701
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             758..784
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             788..808
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             818..3766
                     /codon_start=1
                     /product="Cas9 endonuclease from the Campylobacter jejuni 
                     Type II CRISPR/Cas system (Kim et al., 2016)"
                     /label=CjCas9
                     /translation="ARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLAL
                     PRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELR
                     FRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGE
                     YLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKFE
                     EEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNTE
                     GILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIKA
                     LGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKA
                     LKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKEY
                     RKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGLK
                     INSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLV
                     FTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRN
                     LNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWG
                     FSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKRK
                     FFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKALE
                     LGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGEI
                     KDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFETL
                     SKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK"
     CDS             3770..3790
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3809..3857
                     /note="synthetic polyadenylation signal"
     repeat_region   3865..4005
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3865..3994
                     /label=AAV2 ITR
     rep_origin      4080..4535
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4167..4186)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4377..4398
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4552..4571)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4671..4693
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4731..4749)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4817..4921
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4922..4990,4991..5782)
                     /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(5140..5159)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5953..6541
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6442..6461
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6603..6609,1..123)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg tcgggcgacc tttggtcgcc
       61 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
      121 cctgcggcct ctagactcga ggcgttgaca ttgattattg actagttatt aatagtaatc
      181 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      241 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      301 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      361 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      421 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      481 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      541 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      601 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      661 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      721 aagcagagct ctctggctaa ctaccggtgc caccatgtac ccatacgatg ttccagatta
      781 cgcttcgccg aagaaaaagc gcaaggtcga agcgtccgcc cgcatcctcg ctttcgacat
      841 cggaatctct agtatcggat gggccttctc tgaaaacgac gaactgaaag actgcggcgt
      901 gagaatcttc acaaaggttg aaaaccctaa aacaggcgag tctttagctc tgccacgtag
      961 gttggcccgc tccgcccgaa aaaggctggc tcggcggaag gctcgcctca accacttgaa
     1021 gcatttgata gctaatgagt tcaaactgaa ctacgaagat taccagtcct tcgacgagtc
     1081 attggcaaaa gcctacaaag gcagccttat cagtccttat gagttgagat ttcgcgcact
     1141 caacgaactg ctttctaagc aagactttgc tagggtcatt ctgcacatcg caaaacggcg
     1201 aggttatgac gatatcaaga actccgacga taaagaaaag ggagccattc tcaaggcgat
     1261 caaacagaat gaggaaaaat tggcaaacta ccagagtgtg ggcgagtatc tgtataaaga
     1321 gtatttccag aagtttaagg aaaacagcaa ggagtttaca aacgtcagaa ataaaaagga
     1381 gtcttacgag agatgcatcg cgcagtcatt cctcaaagat gagctgaagc tgatatttaa
     1441 gaagcaacgc gaatttggtt tctcattctc taagaagttc gaagaggagg ttctttccgt
     1501 ggcgttttac aagagggcgc tcaaagactt ctcccacctg gttggtaact gtagtttctt
     1561 cacggatgag aagcgagctc ccaaaaattc tcccctggct ttcatgtttg ttgccctgac
     1621 tcggatcatt aacctgctga acaacctgaa aaatactgaa gggatcttgt atacgaagga
     1681 cgacctaaat gcactcctga atgaagtgct caaaaacgga actctaacct ataaacagac
     1741 caagaaatta ctggggctct ctgacgacta cgagttcaag ggcgagaagg gtacttattt
     1801 tatcgaattc aaaaagtata aggagttcat taaagcattg ggggaacaca acctcagcca
     1861 ggacgatctc aatgaaattg ccaaggacat cacgctgatt aaagacgaga taaaactgaa
     1921 aaaggcactg gccaagtatg acctcaacca gaaccagatc gactctctgt ccaagctgga
     1981 gttcaaagac cacctaaaca tatccttcaa agccctgaaa ctggtcaccc ctctaatgct
     2041 cgaaggaaaa aaatacgacg aggcgtgtaa tgaactgaat cttaaggtgg ccatcaatga
     2101 ggataagaag gactttcttc cagcctttaa cgagacatat tacaaagacg aggtcacaaa
     2161 cccggttgtg ctgagggcca taaaagagta tcggaaggtt ctgaatgccc tcctgaagaa
     2221 gtacggcaaa gtgcacaaaa taaatatcga attggctagg gaggtgggga agaaccattc
     2281 tcagcgagca aagatcgaga aagagcagaa tgagaactac aaagccaaga aagacgccga
     2341 actggagtgc gaaaagctgg ggcttaaaat aaacagtaaa aacatcctga aattaagatt
     2401 gttcaaagag caaaaggagt tttgcgccta ctcaggggaa aaaatcaaaa tatcagacct
     2461 gcaggacgag aaaatgctgg agatcgacca catctatccg tatagcaggt catttgacga
     2521 ttcctacatg aacaaagtgc ttgtgtttac caaacagaac caagaaaagc tgaaccaaac
     2581 cccctttgag gctttcggaa acgactcagc caagtggcag aaaatcgaag tcctagccaa
     2641 gaatctgcct acaaaaaaac aaaagaggat tcttgataag aactataagg acaaggaaca
     2701 gaaaaacttt aaagacagga acctgaatga cacgaggtac attgcgcgac tggttctaaa
     2761 ctataccaaa gactacctgg atttcctccc tctgagcgac gacgagaata ctaaactgaa
     2821 tgatacccag aaaggctcaa aggtccacgt tgaggctaag tccgggatgc tgactagcgc
     2881 cctccgccac acgtggggct tcagcgccaa agatcggaat aatcatcttc atcacgctat
     2941 tgatgcagta atcatagcct acgctaacaa cagcatcgtg aaagccttct ccgatttcaa
     3001 gaaagaacag gagtctaata gcgccgagtt gtacgccaag aaaatttccg aattggacta
     3061 taaaaataag agaaaattct tcgaaccctt ctccgggttt cgccaaaagg tcttagataa
     3121 gatcgacgag attttcgttt ccaagcccga aagaaaaaag ccttcagggg cactgcacga
     3181 agagacattc cgcaaggaag aggaatttta ccaatcttac ggtggtaaag agggagttct
     3241 gaaggctctg gagcttggga agatccgcaa ggtaaacggg aaaatcgtga aaaacgggga
     3301 catgttcagg gtggatatct tcaagcacaa aaagaccaac aagttctacg cagtacccat
     3361 ctacactatg gatttcgctt taaaggttct cccaaataag gcggtggctc gatcgaagaa
     3421 aggagagatc aaggactgga tcttaatgga tgaaaattac gagttttgct tctcgctcta
     3481 caaagatagc ctgattctga tccagacaaa agacatgcag gaaccagaat ttgtttatta
     3541 taacgccttc acgagcagta cagtgtccct gattgtgagc aagcatgata acaagttcga
     3601 gactctgtct aagaatcaga aaatcctttt caagaacgcc aacgagaagg aggtcatcgc
     3661 aaagtcaatt ggcatccaaa acctgaaggt gttcgagaaa tacatagtgt ccgcactcgg
     3721 tgaagtaact aaagccgaat ttcgacagcg cgaggatttt aagaaaagcc ccaagaagaa
     3781 gagaaaggtg gaggccagct aagaattcaa taaaagatct ttgttttcat tagatctgtg
     3841 tgttggtttt ttgtgtgcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     3901 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     3961 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     4021 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     4081 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4141 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4201 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4261 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     4321 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4381 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     4441 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4501 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     4561 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     4621 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4681 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     4741 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     4801 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4861 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4921 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4981 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5041 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5101 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5161 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5221 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5281 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5341 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5401 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5461 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5521 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5581 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag
     5641 ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5701 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5761 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5821 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5881 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5941 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6001 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6061 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6121 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6181 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6241 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6301 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6361 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6421 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6481 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6541 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6601 gtcctgcag
//
