LOCUS       sequence-208551-        6118 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV plasmid expressing CjCas9 under control of miniCMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6118)
  TITLE     Alex Hewitt Lab plasmids
REFERENCE   2  (bases 1 to 6118)
  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-miniCMV-CjCas9
FEATURES             Location/Qualifiers
     source          1..6118
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     128..147
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        128..146
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        165..203
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     200..224
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             259..285
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             289..309
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             319..3267
                     /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             3271..3291
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3310..3358
                     /note="synthetic polyadenylation signal"
     repeat_region   3366..3506
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3366..3495
                     /label=AAV2 ITR
     rep_origin      3581..4036
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3668..3687)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3878..3899
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4053..4072)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4172..4194
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4232..4250)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4318..4422
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4423..4491,4492..5283)
                     /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(4641..4660)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5454..6042
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5943..5962
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6104..6118,1..115)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gcgctcgctc gctcactgag gccgcccggg cgtcgggcga cctttggtcg cccggcctca
       61 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctgcggc
      121 ctctagataa tacgactcac tataggggga tccacgtatg tcgaggtagg cgtgtacggt
      181 gggaggccta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg aggtaccgcc
      241 accaccggtg ccaccatgta cccatacgat gttccagatt acgcttcgcc gaagaaaaag
      301 cgcaaggtcg aagcgtccgc ccgcatcctc gctttcgaca tcggaatctc tagtatcgga
      361 tgggccttct ctgaaaacga cgaactgaaa gactgcggcg tgagaatctt cacaaaggtt
      421 gaaaacccta aaacaggcga gtctttagct ctgccacgta ggttggcccg ctccgcccga
      481 aaaaggctgg ctcggcggaa ggctcgcctc aaccacttga agcatttgat agctaatgag
      541 ttcaaactga actacgaaga ttaccagtcc ttcgacgagt cattggcaaa agcctacaaa
      601 ggcagcctta tcagtcctta tgagttgaga tttcgcgcac tcaacgaact gctttctaag
      661 caagactttg ctagggtcat tctgcacatc gcaaaacggc gaggttatga cgatatcaag
      721 aactccgacg ataaagaaaa gggagccatt ctcaaggcga tcaaacagaa tgaggaaaaa
      781 ttggcaaact accagagtgt gggcgagtat ctgtataaag agtatttcca gaagtttaag
      841 gaaaacagca aggagtttac aaacgtcaga aataaaaagg agtcttacga gagatgcatc
      901 gcgcagtcat tcctcaaaga tgagctgaag ctgatattta agaagcaacg cgaatttggt
      961 ttctcattct ctaagaagtt cgaagaggag gttctttccg tggcgtttta caagagggcg
     1021 ctcaaagact tctcccacct ggttggtaac tgtagtttct tcacggatga gaagcgagct
     1081 cccaaaaatt ctcccctggc tttcatgttt gttgccctga ctcggatcat taacctgctg
     1141 aacaacctga aaaatactga agggatcttg tatacgaagg acgacctaaa tgcactcctg
     1201 aatgaagtgc tcaaaaacgg aactctaacc tataaacaga ccaagaaatt actggggctc
     1261 tctgacgact acgagttcaa gggcgagaag ggtacttatt ttatcgaatt caaaaagtat
     1321 aaggagttca ttaaagcatt gggggaacac aacctcagcc aggacgatct caatgaaatt
     1381 gccaaggaca tcacgctgat taaagacgag ataaaactga aaaaggcact ggccaagtat
     1441 gacctcaacc agaaccagat cgactctctg tccaagctgg agttcaaaga ccacctaaac
     1501 atatccttca aagccctgaa actggtcacc cctctaatgc tcgaaggaaa aaaatacgac
     1561 gaggcgtgta atgaactgaa tcttaaggtg gccatcaatg aggataagaa ggactttctt
     1621 ccagccttta acgagacata ttacaaagac gaggtcacaa acccggttgt gctgagggcc
     1681 ataaaagagt atcggaaggt tctgaatgcc ctcctgaaga agtacggcaa agtgcacaaa
     1741 ataaatatcg aattggctag ggaggtgggg aagaaccatt ctcagcgagc aaagatcgag
     1801 aaagagcaga atgagaacta caaagccaag aaagacgccg aactggagtg cgaaaagctg
     1861 gggcttaaaa taaacagtaa aaacatcctg aaattaagat tgttcaaaga gcaaaaggag
     1921 ttttgcgcct actcagggga aaaaatcaaa atatcagacc tgcaggacga gaaaatgctg
     1981 gagatcgacc acatctatcc gtatagcagg tcatttgacg attcctacat gaacaaagtg
     2041 cttgtgttta ccaaacagaa ccaagaaaag ctgaaccaaa ccccctttga ggctttcgga
     2101 aacgactcag ccaagtggca gaaaatcgaa gtcctagcca agaatctgcc tacaaaaaaa
     2161 caaaagagga ttcttgataa gaactataag gacaaggaac agaaaaactt taaagacagg
     2221 aacctgaatg acacgaggta cattgcgcga ctggttctaa actataccaa agactacctg
     2281 gatttcctcc ctctgagcga cgacgagaat actaaactga atgataccca gaaaggctca
     2341 aaggtccacg ttgaggctaa gtccgggatg ctgactagcg ccctccgcca cacgtggggc
     2401 ttcagcgcca aagatcggaa taatcatctt catcacgcta ttgatgcagt aatcatagcc
     2461 tacgctaaca acagcatcgt gaaagccttc tccgatttca agaaagaaca ggagtctaat
     2521 agcgccgagt tgtacgccaa gaaaatttcc gaattggact ataaaaataa gagaaaattc
     2581 ttcgaaccct tctccgggtt tcgccaaaag gtcttagata agatcgacga gattttcgtt
     2641 tccaagcccg aaagaaaaaa gccttcaggg gcactgcacg aagagacatt ccgcaaggaa
     2701 gaggaatttt accaatctta cggtggtaaa gagggagttc tgaaggctct ggagcttggg
     2761 aagatccgca aggtaaacgg gaaaatcgtg aaaaacgggg acatgttcag ggtggatatc
     2821 ttcaagcaca aaaagaccaa caagttctac gcagtaccca tctacactat ggatttcgct
     2881 ttaaaggttc tcccaaataa ggcggtggct cgatcgaaga aaggagagat caaggactgg
     2941 atcttaatgg atgaaaatta cgagttttgc ttctcgctct acaaagatag cctgattctg
     3001 atccagacaa aagacatgca ggaaccagaa tttgtttatt ataacgcctt cacgagcagt
     3061 acagtgtccc tgattgtgag caagcatgat aacaagttcg agactctgtc taagaatcag
     3121 aaaatccttt tcaagaacgc caacgagaag gaggtcatcg caaagtcaat tggcatccaa
     3181 aacctgaagg tgttcgagaa atacatagtg tccgcactcg gtgaagtaac taaagccgaa
     3241 tttcgacagc gcgaggattt taagaaaagc cccaagaaga agagaaaggt ggaggccagc
     3301 taagaattca ataaaagatc tttgttttca ttagatctgt gtgttggttt tttgtgtgcg
     3361 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     3421 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     3481 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     3541 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     3601 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     3661 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3721 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3781 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     3841 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3901 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     3961 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     4021 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     4081 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4141 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     4201 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     4261 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     4321 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     4381 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     4441 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     4501 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     4561 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     4621 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     4681 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     4741 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     4801 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     4861 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     4921 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     4981 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     5041 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     5101 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     5161 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     5221 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     5281 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     5341 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5401 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5461 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5521 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5581 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     5641 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5701 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5761 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5821 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5881 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     5941 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6001 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6061 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtcctgcag gcagctgc
//
