LOCUS       sequence-359730-        7874 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing mammalian codon optimized engineered chimeric 
            PlmCasX-R1, sgRNAv2 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 7874)
  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 7874)
  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: pCAT100
FEATURES             Location/Qualifiers
     source          1..7874
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          127..354
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      366..375
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             378..398
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3363..3383
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3384..3431
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             3441..3497
                     /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             3498..4094
                     /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     3991..4011
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4128..4145)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4134..4341
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4350..4490
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4350..4479
                     /label=AAV2 ITR
     rep_origin      4565..5020
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4652..4671)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4862..4883
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5037..5056)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5156..5178
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5216..5234)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5302..5406
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5407..5475,5476..6267)
                     /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(5625..5644)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6438..7026
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6927..6946
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        7088..7336
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7088..7108
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7259..7278
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        7470..7755
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
       61 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
      121 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
      181 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
      241 tctcctccgg gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt
      301 ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga
      361 ccggtgccac catggcccca aagaagaagc ggaaggtcag catgcaagag atcaagagaa
      421 tcaacaagat cagaaggaga ctggtcaagg acagcaacac aaagaaggcc ggcaagacag
      481 gccccatgaa aaccctgctc gtcagagtga tgacccctga cctgagagag cggctggaaa
      541 acctgagaaa gaagcccgag aacatccctc agcctatcag caacaccagc agggccaacc
      601 tgaacaagct gctgaccgac tacaccgaga tgaagaaagc catcctgcac gtgtactggg
      661 aagagttcca gaaagacccc gtgggcctga tgagcagagt tgctcagccc gctcctaaga
      721 acatcgacca gagaaagctg atccccgtga aggacggcaa cgagagactg acctctagcg
      781 gctttgcctg cagccagtgt tgccagcctc tgtacgtgta caagctggaa caagtgaacg
      841 acaagggcaa gccccacacc aactacttcg gcagatgcaa cgtgtccgag cacgagaggc
      901 tgatcctgct gtctcctcac aagcccgagg ccaacgatga gctggtcaca tacagcctgg
      961 gcaagttcgg acagagagcc ctggacttct acagcatcca cgtgaccagg gagagcaatc
     1021 accctgtgaa gcccctggaa cagatcggcg gcaatagctg tgcctctgga cctgtgggaa
     1081 aagccctgag cgacgcctgt atgggagccg tggcatcctt cctgaccaag taccaggaca
     1141 tcatcctgga acaccagaaa gtgatcaaga agaacgagaa aagactggcc aacctcaagg
     1201 atatcgccag cgctaacggc ctggcctttc ctaagatcac cctgcctcca cagcctcaca
     1261 ccaaagaggg catcgaggcc tacaacaacg tggtggccca gatcgtgatt tgggtcaacc
     1321 tgaatctgtg gcagaagctg aagatcggca gggacgaagc caagccactg cagagactga
     1381 agggcttccc tagcttccct ctggtggaaa gacaggccaa tgaagtggat tggtgggaca
     1441 tggtctgcaa cgtgaagaag ctgatcaacg agaagaaaga ggatggcaag gttttctggc
     1501 agaacctggc cggctacaag agacaagaag ccctgctgcc ttacctgagc agcgaagagg
     1561 accataaaaa gcgggagggg tctcttgaaa acccgaagaa gttcgccaga taccagttcg
     1621 gcgacctgct gctgcacctg gaaaagaagc acggcgagga ctggggcaaa gtgtacgatg
     1681 aggcctggga gagaatcgac aagaaggtgg aaggcctgag caagcacatt aagctggaag
     1741 aggaaagaag gagcgaggac gcccaatcta aagccgctct gaccgattgg ctgagagcca
     1801 aggccagctt tgtgatcgag ggcctgaaag aggccgacaa ggacgagttc tgcagatgcg
     1861 agctgaagct gcagaagtgg tacggcgatc tgagaggcaa gcccttcgcc attgaggccg
     1921 agaacagcat cctggacatc agcggcttca gcaagcagta caactgcgcc ttcatttggc
     1981 agaaagacgg cgtcaagaaa ctgaacctgt acctgatcat caattacttc aaaggcggca
     2041 agctgcggtt caagaagatc aaacccgagg ccttcgaggc taacagattc tacaccgtga
     2101 tcaacaaaaa gtccggcgag atcgtgccca tggaagtgaa cttcaacttc gacgacccca
     2161 acctgattat cctgcctctg gccttcggca agagacaggg cagagagttc atctggaacg
     2221 atctgctgag cctggaaacc ggctctctga agctggccaa tggcagagtg atcgagaaaa
     2281 ccctgtacaa caggagaacc agacaggacg agcctgctct gtttgtggcc ctgaccttcg
     2341 agagaagaga ggtgctggac agcagcaaca tcaagcccat gaacctgatc ggcatcgacc
     2401 ggggcgagaa tatccctgct gtgatcgccc tgacagaccc tgaaggatgc ccactgagca
     2461 gattcaagga ctccctgggc aaccctacac acatcctgag aatcggcgag agctacaaag
     2521 agaagcagag gacaatccag gccgccaaag aggtggaaca gagaagagcc ggcggatact
     2581 ctaggaagta cgccagcaag gccaagaatc tggccgacga catggtccga aacaccgcca
     2641 gagatctgct gtactacgcc gtgacacagg acgccatgct gatcttcgag aatctgagca
     2701 gaggcttcgg ccggcagggc aagagaacct ttatggccga gaggcagtac accagaatgg
     2761 aagattggct cacagctaaa ctggcctacg agggactgcc cagcaagacc tacctgtcca
     2821 aaacactggc ccagtatacc tccaagacct gcagcaattg cggcttcacc atcaccagcg
     2881 ccgactacga cagagtgctg gaaaagctca agaaaaccgc caccggctgg atgaccacca
     2941 tcaacggcaa agagctgaag gttgagggcc agatcaccta ctacaacagg tacaagaggc
     3001 agaacgtcgt gaaggatctg agcgtggaac tggacagact gagcgaagag agcgtgaaca
     3061 acgacatcag cagctggaca aagggcagat caggcgaggc tctgagcctg ctgaagaaga
     3121 ggtttagcca cagacctgtg caagagaagt tcgtgtgcct gaactgcggc ttcgagacac
     3181 acgccgatga acaggctgcc ctgaacattg ccagaagctg gctgttcctg agaagccaag
     3241 agtacaagaa gtaccagacc aacaagacca ccggcaacac cgacaagagg gcctttgtgg
     3301 aaacctggca gagcttctac agaaaaaagc tgaaagaagt ctggaagccc gccgtgacta
     3361 gtccaaaaaa gaagagaaag gtagattaca aagatgacga tgacaaagac tacaaggatg
     3421 atgatgataa gggatccggc gcaacaaact tctctctgct gaaacaagcc ggagatgtcg
     3481 aagagaatcc tggaccgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
     3541 tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
     3601 ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
     3661 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct
     3721 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
     3781 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
     3841 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg
     3901 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
     3961 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct
     4021 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
     4081 agcccggtgc ctgaacgcgt taagaattcc tagagctcgc tgatcagcct cgactgtgcc
     4141 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4201 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4261 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4321 gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc
     4381 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     4441 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     4501 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     4561 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4621 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
     4681 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4741 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     4801 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     4861 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
     4921 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
     4981 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     5041 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     5101 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5161 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     5221 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     5281 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5341 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5401 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5461 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5521 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5581 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5641 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5701 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5761 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     5821 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     5881 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     5941 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     6001 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     6061 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     6121 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6181 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6241 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6301 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6361 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6421 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6481 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6541 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6601 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6661 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6721 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6781 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6841 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6901 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6961 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     7021 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     7081 cacatgtgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     7141 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     7201 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     7261 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     7321 gaaaggacga aacaccggcg cttttatctc attactttga gagccatcac cagcgactat
     7381 gtcgtatggg taaagcgctt atttatcgga gaaaccgata aataagaagc atcaaagggg
     7441 tcttcgagaa gacccttttt tttggtaccc gttacataac ttacggtaaa tggcccgcct
     7501 ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc
     7561 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     7621 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     7681 tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     7741 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
     7801 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
     7861 gcgggggggg gggg
//
