LOCUS       sequence-359727-        8721 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing mammalian codon optimized engineered chimeric 
            PlmCasX-R1, mNeonGreen, 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 8721)
  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 8721)
  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: pCAT527
FEATURES             Location/Qualifiers
     source          1..8721
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     482..501
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        643..891
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     643..663
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     814..833
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        1025..1310
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     gap             1431..1507
                     /estimated_length=77
     intron          1629..1856
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1868..1877
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1880..1900
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4865..4885
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4886..4933
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             4943..4999
                     /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             5717..5773
                     /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             5774..6370
                     /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     6267..6287
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6404..6421)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6410..6617
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6626..6766
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6626..6755
                     /label=AAV2 ITR
     rep_origin      6841..7296
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6928..6947)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7138..7159
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7313..7332)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7432..7454
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7492..7510)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7578..7682
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7683..7751,7752..8543)
                     /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(7901..7920)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(8714..8721,1..581)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
       61 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
      121 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
      181 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
      241 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
      301 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
      361 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
      421 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
      481 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
      541 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
      601 ttttacggtt cctggccttt tgctggcctt ttgctcacat gtgagggcct atttcccatg
      661 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
      721 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
      781 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
      841 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cggcgctttt
      901 atctcattac tttgagagcc atcaccagcg actatgtcgt atgggtaaag cgcttattta
      961 tcggagaaac cgataaataa gaagcatcaa aggggtcttc gagaagaccc ttttttttgg
     1021 tacccgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     1081 cattgacgtc aatagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     1141 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     1201 ttgacgtcaa tgacggtaaa tggcccgcct ggcattgtgc ccagtacatg accttatggg
     1261 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtcgaggtga
     1321 gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt
     1381 tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg nnnnnnnnnn
     1441 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
     1501 nnnnnnnggg cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc
     1561 gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc
     1621 ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc
     1681 gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc
     1741 cttctcctcc gggctgtaat tagctgagca agaggtaagg gtttaaggga tggttggttg
     1801 gtggggtatt aatgtttaat tacctggagc acctgcctga aatcactttt tttcaggttg
     1861 gaccggtgcc accatggccc caaagaagaa gcggaaggtc agcatgcaag agatcaagag
     1921 aatcaacaag atcagaagga gactggtcaa ggacagcaac acaaagaagg ccggcaagac
     1981 aggccccatg aaaaccctgc tcgtcagagt gatgacccct gacctgagag agcggctgga
     2041 aaacctgaga aagaagcccg agaacatccc tcagcctatc agcaacacca gcagggccaa
     2101 cctgaacaag ctgctgaccg actacaccga gatgaagaaa gccatcctgc acgtgtactg
     2161 ggaagagttc cagaaagacc ccgtgggcct gatgagcaga gttgctcagc ccgctcctaa
     2221 gaacatcgac cagagaaagc tgatccccgt gaaggacggc aacgagagac tgacctctag
     2281 cggctttgcc tgcagccagt gttgccagcc tctgtacgtg tacaagctgg aacaagtgaa
     2341 cgacaagggc aagccccaca ccaactactt cggcagatgc aacgtgtccg agcacgagag
     2401 gctgatcctg ctgtctcctc acaagcccga ggccaacgat gagctggtca catacagcct
     2461 gggcaagttc ggacagagag ccctggactt ctacagcatc cacgtgacca gggagagcaa
     2521 tcaccctgtg aagcccctgg aacagatcgg cggcaatagc tgtgcctctg gacctgtggg
     2581 aaaagccctg agcgacgcct gtatgggagc cgtggcatcc ttcctgacca agtaccagga
     2641 catcatcctg gaacaccaga aagtgatcaa gaagaacgag aaaagactgg ccaacctcaa
     2701 ggatatcgcc agcgctaacg gcctggcctt tcctaagatc accctgcctc cacagcctca
     2761 caccaaagag ggcatcgagg cctacaacaa cgtggtggcc cagatcgtga tttgggtcaa
     2821 cctgaatctg tggcagaagc tgaagatcgg cagggacgaa gccaagccac tgcagagact
     2881 gaagggcttc cctagcttcc ctctggtgga aagacaggcc aatgaagtgg attggtggga
     2941 catggtctgc aacgtgaaga agctgatcaa cgagaagaaa gaggatggca aggttttctg
     3001 gcagaacctg gccggctaca agagacaaga agccctgctg ccttacctga gcagcgaaga
     3061 ggaccataaa aagcgggagg ggtctcttga aaacccgaag aagttcgcca gataccagtt
     3121 cggcgacctg ctgctgcacc tggaaaagaa gcacggcgag gactggggca aagtgtacga
     3181 tgaggcctgg gagagaatcg acaagaaggt ggaaggcctg agcaagcaca ttaagctgga
     3241 agaggaaaga aggagcgagg acgcccaatc taaagccgct ctgaccgatt ggctgagagc
     3301 caaggccagc tttgtgatcg agggcctgaa agaggccgac aaggacgagt tctgcagatg
     3361 cgagctgaag ctgcagaagt ggtacggcga tctgagaggc aagcccttcg ccattgaggc
     3421 cgagaacagc atcctggaca tcagcggctt cagcaagcag tacaactgcg ccttcatttg
     3481 gcagaaagac ggcgtcaaga aactgaacct gtacctgatc atcaattact tcaaaggcgg
     3541 caagctgcgg ttcaagaaga tcaaacccga ggccttcgag gctaacagat tctacaccgt
     3601 gatcaacaaa aagtccggcg agatcgtgcc catggaagtg aacttcaact tcgacgaccc
     3661 caacctgatt atcctgcctc tggccttcgg caagagacag ggcagagagt tcatctggaa
     3721 cgatctgctg agcctggaaa ccggctctct gaagctggcc aatggcagag tgatcgagaa
     3781 aaccctgtac aacaggagaa ccagacagga cgagcctgct ctgtttgtgg ccctgacctt
     3841 cgagagaaga gaggtgctgg acagcagcaa catcaagccc atgaacctga tcggcatcga
     3901 ccggggcgag aatatccctg ctgtgatcgc cctgacagac cctgaaggat gcccactgag
     3961 cagattcaag gactccctgg gcaaccctac acacatcctg agaatcggcg agagctacaa
     4021 agagaagcag aggacaatcc aggccgccaa agaggtggaa cagagaagag ccggcggata
     4081 ctctaggaag tacgccagca aggccaagaa tctggccgac gacatggtcc gaaacaccgc
     4141 cagagatctg ctgtactacg ccgtgacaca ggacgccatg ctgatcttcg agaatctgag
     4201 cagaggcttc ggccggcagg gcaagagaac ctttatggcc gagaggcagt acaccagaat
     4261 ggaagattgg ctcacagcta aactggccta cgagggactg cccagcaaga cctacctgtc
     4321 caaaacactg gcccagtata cctccaagac ctgcagcaat tgcggcttca ccatcaccag
     4381 cgccgactac gacagagtgc tggaaaagct caagaaaacc gccaccggct ggatgaccac
     4441 catcaacggc aaagagctga aggttgaggg ccagatcacc tactacaaca ggtacaagag
     4501 gcagaacgtc gtgaaggatc tgagcgtgga actggacaga ctgagcgaag agagcgtgaa
     4561 caacgacatc agcagctgga caaagggcag atcaggcgag gctctgagcc tgctgaagaa
     4621 gaggtttagc cacagacctg tgcaagagaa gttcgtgtgc ctgaactgcg gcttcgagac
     4681 acacgccgat gaacaggctg ccctgaacat tgccagaagc tggctgttcc tgagaagcca
     4741 agagtacaag aagtaccaga ccaacaagac caccggcaac accgacaaga gggcctttgt
     4801 ggaaacctgg cagagcttct acagaaaaaa gctgaaagaa gtctggaagc ccgccgtgac
     4861 tagtccaaaa aagaagagaa aggtagatta caaagatgac gatgacaaag actacaagga
     4921 tgatgatgat aagggatccg gcgccaccaa cttcagcctg ctgaagcagg ccggcgacgt
     4981 ggaggagaac cccggcccca tggtaagtaa gggcgaagag gataatatgg cttctttgcc
     5041 cgccacacac gaactgcaca tattcggatc aataaacggg gtggatttcg atatggtcgg
     5101 ccagggaaca ggaaacccta atgacggcta cgaagagctc aatctcaaat ctaccaaagg
     5161 ggacttacaa ttctctccat ggatcctagt gccacatatt ggatatggct tccatcaata
     5221 ccttccatat ccagacggta tgagcccctt ccaggctgca atggtggacg ggtctggata
     5281 tcaggttcac agaacaatgc aatttgagga cggtgcatcc cttacagtta attacaggta
     5341 cacatacgaa ggaagtcaca tcaaagggga ggcgcaggtg aagggcaccg ggttcccagc
     5401 tgatggacct gtaatgacaa attcactcac agccgccgat tggtgcaggt caaagaaaac
     5461 ctatcctaat gacaaaacaa ttatatccac atttaaatgg tcttatacca caggaaatgg
     5521 aaagagatac agatctacag ctagaacaac ttacacattt gctaaaccaa tggcagccaa
     5581 ctaccttaag aatcaaccaa tgtatgtgtt cagaaagact gaactaaaac acagtaaaac
     5641 agaacttaat ttcaaagagt ggcagaaggc cttcacagac gttatgggta tggatgaact
     5701 gtacaaggga tccggcgcaa caaacttctc tctgctgaaa caagccggag atgtcgaaga
     5761 gaatcctgga ccgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     5821 cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     5881 cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     5941 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac
     6001 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     6061 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     6121 gcccaaggag cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc accagggcaa
     6181 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     6241 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     6301 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
     6361 cggtgcctga acgcgttaag aattcctaga gctcgctgat cagcctcgac tgtgccttct
     6421 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     6481 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     6541 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat
     6601 agcaggcatg ctggggagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     6661 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     6721 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     6781 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     6841 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     6901 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     6961 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     7021 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     7081 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     7141 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
     7201 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
     7261 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     7321 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     7381 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     7441 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     7501 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     7561 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     7621 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     7681 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     7741 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     7801 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     7861 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     7921 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     7981 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     8041 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     8101 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     8161 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     8221 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     8281 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     8341 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa
     8401 gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     8461 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     8521 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     8581 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     8641 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     8701 tcaaaggatc ttcttgagat c
//
