LOCUS       sequence-359728-        8639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing mammalian codon optimized wt PlmCasX, mNeonGreen,
            sgRNAv1 scaffold, and restriction sites to clone in new spacers.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8639)
  AUTHORS   Tsuchida CA, Zhang S, Doost MS, Zhao Y, Wang J, O'Brien E, Fang H, 
            Li CP, Li D, Hai ZY, Chuck J, Brotzmann J, Vartoumian A, Burstein D,
            Chen XW, Nogales E, Doudna JA, Liu JG
  TITLE     Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome 
            editing activity.
  JOURNAL   Mol Cell. 2022 Mar 17;82(6):1199-1209.e6. doi: 
            10.1016/j.molcel.2022.02.002. Epub 2022 Feb 25.
  PUBMED    35219382
REFERENCE   2  (bases 1 to 8639)
  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.molcel.2022.02.002"; journalName: "Mol Cell"; date: 
            "2022-03-17- 17"; volume: "82"; issue: "6"; pages: "1199-1209.e6"
COMMENT     Sequence Label: pCAT526
FEATURES             Location/Qualifiers
     source          1..8639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          106..333
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      345..354
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             357..377
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3321..3341
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3342..3389
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             3399..3455
                     /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"
     CDS             4173..4229
                     /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"
     CDS             4230..4826
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     4723..4743
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4860..4877)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4866..5073
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5082..5222
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5082..5211
                     /label=AAV2 ITR
     rep_origin      5297..5752
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5384..5403)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5594..5615
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5769..5788)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5888..5910
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5948..5966)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6034..6138
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6139..6207,6208..6999)
                     /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(6357..6376)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7170..7758
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7659..7678
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        7820..8068
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7820..7840
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7991..8010
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        8209..8494
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     gap             8606..8628
                     /estimated_length=23
ORIGIN
        1 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
       61 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc
      121 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
      181 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
      241 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
      301 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggccccaa
      361 agaagaagcg gaaggtcagc atgcaagaga tcaagagaat caacaagatc agaaggagac
      421 tggtcaagga cagcaacaca aagaaggccg gcaagacagg ccccatgaaa accctgctcg
      481 tcagagtgat gacccctgac ctgagagagc ggctggaaaa cctgagaaag aagcccgaga
      541 acatccctca gcctatcagc aacaccagca gggccaacct gaacaagctg ctgaccgact
      601 acaccgagat gaagaaagcc atcctgcacg tgtactggga agagttccag aaagaccccg
      661 tgggcctgat gagcagagtt gctcagcccg ctcctaagaa catcgaccag agaaagctga
      721 tccccgtgaa ggacggcaac gagagactga cctctagcgg ctttgcctgc agccagtgtt
      781 gccagcctct gtacgtgtac aagctggaac aagtgaacga caagggcaag ccccacacca
      841 actacttcgg cagatgcaac gtgtccgagc acgagaggct gatcctgctg tctcctcaca
      901 agcccgaggc caacgatgag ctggtcacat acagcctggg caagttcgga cagagagccc
      961 tggacttcta cagcatccac gtgaccaggg agagcaatca ccctgtgaag cccctggaac
     1021 agatcggcgg caatagctgt gcctctggac ctgtgggaaa agccctgagc gacgcctgta
     1081 tgggagccgt ggcatccttc ctgaccaagt accaggacat catcctggaa caccagaaag
     1141 tgatcaagaa gaacgagaaa agactggcca acctcaagga tatcgccagc gctaacggcc
     1201 tggcctttcc taagatcacc ctgcctccac agcctcacac caaagagggc atcgaggcct
     1261 acaacaacgt ggtggcccag atcgtgattt gggtcaacct gaatctgtgg cagaagctga
     1321 agatcggcag ggacgaagcc aagccactgc agagactgaa gggcttccct agcttccctc
     1381 tggtggaaag acaggccaat gaagtggatt ggtgggacat ggtctgcaac gtgaagaagc
     1441 tgatcaacga gaagaaagag gatggcaagg ttttctggca gaacctggcc ggctacaaga
     1501 gacaagaagc cctgctgcct tacctgagca gcgaagagga ccggaagaag ggcaagaagt
     1561 tcgccagata ccagttcggc gacctgctgc tgcacctgga aaagaagcac ggcgaggact
     1621 ggggcaaagt gtacgatgag gcctgggaga gaatcgacaa gaaggtggaa ggcctgagca
     1681 agcacattaa gctggaagag gaaagaagga gcgaggacgc ccaatctaaa gccgctctga
     1741 ccgattggct gagagccaag gccagctttg tgatcgaggg cctgaaagag gccgacaagg
     1801 acgagttctg cagatgcgag ctgaagctgc agaagtggta cggcgatctg agaggcaagc
     1861 ccttcgccat tgaggccgag aacagcatcc tggacatcag cggcttcagc aagcagtaca
     1921 actgcgcctt catttggcag aaagacggcg tcaagaaact gaacctgtac ctgatcatca
     1981 attacttcaa aggcggcaag ctgcggttca agaagatcaa acccgaggcc ttcgaggcta
     2041 acagattcta caccgtgatc aacaaaaagt ccggcgagat cgtgcccatg gaagtgaact
     2101 tcaacttcga cgaccccaac ctgattatcc tgcctctggc cttcggcaag agacagggca
     2161 gagagttcat ctggaacgat ctgctgagcc tggaaaccgg ctctctgaag ctggccaatg
     2221 gcagagtgat cgagaaaacc ctgtacaaca ggagaaccag acaggacgag cctgctctgt
     2281 ttgtggccct gaccttcgag agaagagagg tgctggacag cagcaacatc aagcccatga
     2341 acctgatcgg catcgaccgg ggcgagaata tccctgctgt gatcgccctg acagaccctg
     2401 aaggatgccc actgagcaga ttcaaggact ccctgggcaa ccctacacac atcctgagaa
     2461 tcggcgagag ctacaaagag aagcagagga caatccaggc cgccaaagag gtggaacaga
     2521 gaagagccgg cggatactct aggaagtacg ccagcaaggc caagaatctg gccgacgaca
     2581 tggtccgaaa caccgccaga gatctgctgt actacgccgt gacacaggac gccatgctga
     2641 tcttcgagaa tctgagcaga ggcttcggcc ggcagggcaa gagaaccttt atggccgaga
     2701 ggcagtacac cagaatggaa gattggctca cagctaaact ggcctacgag ggactgccca
     2761 gcaagaccta cctgtccaaa acactggccc agtatacctc caagacctgc agcaattgcg
     2821 gcttcaccat caccagcgcc gactacgaca gagtgctgga aaagctcaag aaaaccgcca
     2881 ccggctggat gaccaccatc aacggcaaag agctgaaggt tgagggccag atcacctact
     2941 acaacaggta caagaggcag aacgtcgtga aggatctgag cgtggaactg gacagactga
     3001 gcgaagagag cgtgaacaac gacatcagca gctggacaaa gggcagatca ggcgaggctc
     3061 tgagcctgct gaagaagagg tttagccaca gacctgtgca agagaagttc gtgtgcctga
     3121 actgcggctt cgagacacac gccgatgaac aggctgccct gaacattgcc agaagctggc
     3181 tgttcctgag aagccaagag tacaagaagt accagaccaa caagaccacc ggcaacaccg
     3241 acaagagggc ctttgtggaa acctggcaga gcttctacag aaaaaagctg aaagaagtct
     3301 ggaagcccgc cgtgactagt ccaaaaaaga agagaaaggt agattacaaa gatgacgatg
     3361 acaaagacta caaggatgat gatgataagg gatccggcgc caccaacttc agcctgctga
     3421 agcaggccgg cgacgtggag gagaaccccg gccccatggt aagtaagggc gaagaggata
     3481 atatggcttc tttgcccgcc acacacgaac tgcacatatt cggatcaata aacggggtgg
     3541 atttcgatat ggtcggccag ggaacaggaa accctaatga cggctacgaa gagctcaatc
     3601 tcaaatctac caaaggggac ttacaattct ctccatggat cctagtgcca catattggat
     3661 atggcttcca tcaatacctt ccatatccag acggtatgag ccccttccag gctgcaatgg
     3721 tggacgggtc tggatatcag gttcacagaa caatgcaatt tgaggacggt gcatccctta
     3781 cagttaatta caggtacaca tacgaaggaa gtcacatcaa aggggaggcg caggtgaagg
     3841 gcaccgggtt cccagctgat ggacctgtaa tgacaaattc actcacagcc gccgattggt
     3901 gcaggtcaaa gaaaacctat cctaatgaca aaacaattat atccacattt aaatggtctt
     3961 ataccacagg aaatggaaag agatacagat ctacagctag aacaacttac acatttgcta
     4021 aaccaatggc agccaactac cttaagaatc aaccaatgta tgtgttcaga aagactgaac
     4081 taaaacacag taaaacagaa cttaatttca aagagtggca gaaggccttc acagacgtta
     4141 tgggtatgga tgaactgtac aagggatccg gcgcaacaaa cttctctctg ctgaaacaag
     4201 ccggagatgt cgaagagaat cctggaccga ccgagtacaa gcccacggtg cgcctcgcca
     4261 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     4321 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     4381 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     4441 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     4501 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     4561 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc
     4621 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     4681 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     4741 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     4801 gcatgacccg caagcccggt gcctgaacgc gttaagaatt cctagagctc gctgatcagc
     4861 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     4921 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     4981 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     5041 ggattgggaa gagaatagca ggcatgctgg ggagcggccg caggaacccc tagtgatgga
     5101 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     5161 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     5221 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     5281 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     5341 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc
     5401 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     5461 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     5521 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5581 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc
     5641 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg
     5701 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     5761 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     5821 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5881 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     5941 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     6001 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     6061 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     6121 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     6181 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     6241 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     6301 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     6361 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     6421 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     6481 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6541 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6601 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6661 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6721 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6781 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6841 gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6901 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6961 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     7021 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     7081 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     7141 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     7201 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     7261 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     7321 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     7381 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7441 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7501 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7561 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7621 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7681 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7741 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7801 tggccttttg ctcacatgtg agggcctatt tcccatgatt ccttcatatt tgcatatacg
     7861 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
     7921 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     7981 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     8041 atatatcttg tggaaaggac gaaacaccac atctggcgcg tttattccat tactttggag
     8101 ccagtcccag cgactatgtc gtatggacga agcgcttatt tatcggagag aaaccgataa
     8161 gtaaaacgca tcaaaggggt cttcgagaag accctttttt ttggtacccg ttacataact
     8221 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaatagt
     8281 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     8341 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     8401 taaatggccc gcctggcatt gtgcccagta catgacctta tgggactttc ctacttggca
     8461 gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt
     8521 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt
     8581 attttgtgca gcgakggggg cggggnnnnn nnnnnnnnnn nnnnnnnngg gcgaggcgg
//
