LOCUS       sequence-351032-        8669 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 8669)
  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 8669)
  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..8669
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        651..899
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     651..671
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     822..841
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        1040..1325
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1327..1604
                     /label=chicken beta-actin promoter
     intron          1605..1833
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1845..1854
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1857..1877
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4821..4841
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4842..4889
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             4899..4955
                     /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             5673..5729
                     /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             5730..6326
                     /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     6223..6243
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6360..6377)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6366..6573
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6582..6722
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6582..6711
                     /label=AAV2 ITR
     rep_origin      6797..7252
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6884..6903)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7094..7115
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7269..7288)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7388..7410
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7448..7466)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7534..7638
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7639..7707,7708..8499)
                     /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(7857..7876)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt gagggcctat
      661 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
      721 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
      781 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
      841 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacacca
      901 catctggcgc gtttattcca ttactttgga gccagtccca gcgactatgt cgtatggacg
      961 aagcgcttat ttatcggaga gaaaccgata agtaaaacgc atcaaagggg tcttcgagaa
     1021 gacccttttt tttggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     1081 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
     1141 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     1201 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
     1261 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     1321 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
     1381 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
     1441 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
     1501 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
     1561 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc
     1621 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
     1681 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     1741 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
     1801 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggccccaa
     1861 agaagaagcg gaaggtcagc atgcaagaga tcaagagaat caacaagatc agaaggagac
     1921 tggtcaagga cagcaacaca aagaaggccg gcaagacagg ccccatgaaa accctgctcg
     1981 tcagagtgat gacccctgac ctgagagagc ggctggaaaa cctgagaaag aagcccgaga
     2041 acatccctca gcctatcagc aacaccagca gggccaacct gaacaagctg ctgaccgact
     2101 acaccgagat gaagaaagcc atcctgcacg tgtactggga agagttccag aaagaccccg
     2161 tgggcctgat gagcagagtt gctcagcccg ctcctaagaa catcgaccag agaaagctga
     2221 tccccgtgaa ggacggcaac gagagactga cctctagcgg ctttgcctgc agccagtgtt
     2281 gccagcctct gtacgtgtac aagctggaac aagtgaacga caagggcaag ccccacacca
     2341 actacttcgg cagatgcaac gtgtccgagc acgagaggct gatcctgctg tctcctcaca
     2401 agcccgaggc caacgatgag ctggtcacat acagcctggg caagttcgga cagagagccc
     2461 tggacttcta cagcatccac gtgaccaggg agagcaatca ccctgtgaag cccctggaac
     2521 agatcggcgg caatagctgt gcctctggac ctgtgggaaa agccctgagc gacgcctgta
     2581 tgggagccgt ggcatccttc ctgaccaagt accaggacat catcctggaa caccagaaag
     2641 tgatcaagaa gaacgagaaa agactggcca acctcaagga tatcgccagc gctaacggcc
     2701 tggcctttcc taagatcacc ctgcctccac agcctcacac caaagagggc atcgaggcct
     2761 acaacaacgt ggtggcccag atcgtgattt gggtcaacct gaatctgtgg cagaagctga
     2821 agatcggcag ggacgaagcc aagccactgc agagactgaa gggcttccct agcttccctc
     2881 tggtggaaag acaggccaat gaagtggatt ggtgggacat ggtctgcaac gtgaagaagc
     2941 tgatcaacga gaagaaagag gatggcaagg ttttctggca gaacctggcc ggctacaaga
     3001 gacaagaagc cctgctgcct tacctgagca gcgaagagga ccggaagaag ggcaagaagt
     3061 tcgccagata ccagttcggc gacctgctgc tgcacctgga aaagaagcac ggcgaggact
     3121 ggggcaaagt gtacgatgag gcctgggaga gaatcgacaa gaaggtggaa ggcctgagca
     3181 agcacattaa gctggaagag gaaagaagga gcgaggacgc ccaatctaaa gccgctctga
     3241 ccgattggct gagagccaag gccagctttg tgatcgaggg cctgaaagag gccgacaagg
     3301 acgagttctg cagatgcgag ctgaagctgc agaagtggta cggcgatctg agaggcaagc
     3361 ccttcgccat tgaggccgag aacagcatcc tggacatcag cggcttcagc aagcagtaca
     3421 actgcgcctt catttggcag aaagacggcg tcaagaaact gaacctgtac ctgatcatca
     3481 attacttcaa aggcggcaag ctgcggttca agaagatcaa acccgaggcc ttcgaggcta
     3541 acagattcta caccgtgatc aacaaaaagt ccggcgagat cgtgcccatg gaagtgaact
     3601 tcaacttcga cgaccccaac ctgattatcc tgcctctggc cttcggcaag agacagggca
     3661 gagagttcat ctggaacgat ctgctgagcc tggaaaccgg ctctctgaag ctggccaatg
     3721 gcagagtgat cgagaaaacc ctgtacaaca ggagaaccag acaggacgag cctgctctgt
     3781 ttgtggccct gaccttcgag agaagagagg tgctggacag cagcaacatc aagcccatga
     3841 acctgatcgg catcgaccgg ggcgagaata tccctgctgt gatcgccctg acagaccctg
     3901 aaggatgccc actgagcaga ttcaaggact ccctgggcaa ccctacacac atcctgagaa
     3961 tcggcgagag ctacaaagag aagcagagga caatccaggc cgccaaagag gtggaacaga
     4021 gaagagccgg cggatactct aggaagtacg ccagcaaggc caagaatctg gccgacgaca
     4081 tggtccgaaa caccgccaga gatctgctgt actacgccgt gacacaggac gccatgctga
     4141 tcttcgagaa tctgagcaga ggcttcggcc ggcagggcaa gagaaccttt atggccgaga
     4201 ggcagtacac cagaatggaa gattggctca cagctaaact ggcctacgag ggactgccca
     4261 gcaagaccta cctgtccaaa acactggccc agtatacctc caagacctgc agcaattgcg
     4321 gcttcaccat caccagcgcc gactacgaca gagtgctgga aaagctcaag aaaaccgcca
     4381 ccggctggat gaccaccatc aacggcaaag agctgaaggt tgagggccag atcacctact
     4441 acaacaggta caagaggcag aacgtcgtga aggatctgag cgtggaactg gacagactga
     4501 gcgaagagag cgtgaacaac gacatcagca gctggacaaa gggcagatca ggcgaggctc
     4561 tgagcctgct gaagaagagg tttagccaca gacctgtgca agagaagttc gtgtgcctga
     4621 actgcggctt cgagacacac gccgatgaac aggctgccct gaacattgcc agaagctggc
     4681 tgttcctgag aagccaagag tacaagaagt accagaccaa caagaccacc ggcaacaccg
     4741 acaagagggc ctttgtggaa acctggcaga gcttctacag aaaaaagctg aaagaagtct
     4801 ggaagcccgc cgtgactagt ccaaaaaaga agagaaaggt agattacaaa gatgacgatg
     4861 acaaagacta caaggatgat gatgataagg gatccggcgc caccaacttc agcctgctga
     4921 agcaggccgg cgacgtggag gagaaccccg gccccatggt aagtaagggc gaagaggata
     4981 atatggcttc tttgcccgcc acacacgaac tgcacatatt cggatcaata aacggggtgg
     5041 atttcgatat ggtcggccag ggaacaggaa accctaatga cggctacgaa gagctcaatc
     5101 tcaaatctac caaaggggac ttacaattct ctccatggat cctagtgcca catattggat
     5161 atggcttcca tcaatacctt ccatatccag acggtatgag ccccttccag gctgcaatgg
     5221 tggacgggtc tggatatcag gttcacagaa caatgcaatt tgaggacggt gcatccctta
     5281 cagttaatta caggtacaca tacgaaggaa gtcacatcaa aggggaggcg caggtgaagg
     5341 gcaccgggtt cccagctgat ggacctgtaa tgacaaattc actcacagcc gccgattggt
     5401 gcaggtcaaa gaaaacctat cctaatgaca aaacaattat atccacattt aaatggtctt
     5461 ataccacagg aaatggaaag agatacagat ctacagctag aacaacttac acatttgcta
     5521 aaccaatggc agccaactac cttaagaatc aaccaatgta tgtgttcaga aagactgaac
     5581 taaaacacag taaaacagaa cttaatttca aagagtggca gaaggccttc acagacgtta
     5641 tgggtatgga tgaactgtac aagggatccg gcgcaacaaa cttctctctg ctgaaacaag
     5701 ccggagatgt cgaagagaat cctggaccga ccgagtacaa gcccacggtg cgcctcgcca
     5761 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     5821 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     5881 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     5941 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     6001 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     6061 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc
     6121 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     6181 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     6241 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     6301 gcatgacccg caagcccggt gcctgaacgc gttaagaatt cctagagctc gctgatcagc
     6361 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     6421 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     6481 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     6541 ggattgggaa gagaatagca ggcatgctgg ggagcggccg caggaacccc tagtgatgga
     6601 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     6661 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     6721 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     6781 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     6841 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     6901 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     6961 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     7021 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     7081 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc
     7141 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     7201 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     7261 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     7321 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     7381 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     7441 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     7501 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     7561 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     7621 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     7681 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     7741 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     7801 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     7861 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     7921 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     7981 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     8041 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     8101 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     8161 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     8221 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     8281 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     8341 gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     8401 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     8461 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     8521 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     8581 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     8641 gaccccgtag aaaagatcaa aggatcttc
//
