LOCUS       sequence-351033-        8683 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 8683)
  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 8683)
  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..8683
                     /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        1033..1318
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1320..1597
                     /label=chicken beta-actin promoter
     intron          1598..1826
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1838..1847
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1850..1870
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4835..4855
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4856..4903
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             4913..4969
                     /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             5687..5743
                     /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             5744..6340
                     /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     6237..6257
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6374..6391)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6380..6587
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6596..6736
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6596..6725
                     /label=AAV2 ITR
     rep_origin      6811..7266
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6898..6917)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7108..7129
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7283..7302)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7402..7424
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7462..7480)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7548..7652
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7653..7721,7722..8513)
                     /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(7871..7890)
                     /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 cgaaacaccg
      901 gcgcttttat ctcattactt tgagagccat caccagcgac tatgtcgtat gggtaaagcg
      961 cttatttatc ggagaaaccg ataaataaga agcatcaaag gggtcttcga gaagaccctt
     1021 ttttttggta cccgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     1081 cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt
     1141 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     1201 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac
     1261 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     1321 cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat
     1381 tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg
     1441 gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc
     1501 ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg
     1561 gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcga cgctgccttc
     1621 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
     1681 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca
     1741 agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc
     1801 acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggccc caaagaagaa
     1861 gcggaaggtc agcatgcaag agatcaagag aatcaacaag atcagaagga gactggtcaa
     1921 ggacagcaac acaaagaagg ccggcaagac aggccccatg aaaaccctgc tcgtcagagt
     1981 gatgacccct gacctgagag agcggctgga aaacctgaga aagaagcccg agaacatccc
     2041 tcagcctatc agcaacacca gcagggccaa cctgaacaag ctgctgaccg actacaccga
     2101 gatgaagaaa gccatcctgc acgtgtactg ggaagagttc cagaaagacc ccgtgggcct
     2161 gatgagcaga gttgctcagc ccgctcctaa gaacatcgac cagagaaagc tgatccccgt
     2221 gaaggacggc aacgagagac tgacctctag cggctttgcc tgcagccagt gttgccagcc
     2281 tctgtacgtg tacaagctgg aacaagtgaa cgacaagggc aagccccaca ccaactactt
     2341 cggcagatgc aacgtgtccg agcacgagag gctgatcctg ctgtctcctc acaagcccga
     2401 ggccaacgat gagctggtca catacagcct gggcaagttc ggacagagag ccctggactt
     2461 ctacagcatc cacgtgacca gggagagcaa tcaccctgtg aagcccctgg aacagatcgg
     2521 cggcaatagc tgtgcctctg gacctgtggg aaaagccctg agcgacgcct gtatgggagc
     2581 cgtggcatcc ttcctgacca agtaccagga catcatcctg gaacaccaga aagtgatcaa
     2641 gaagaacgag aaaagactgg ccaacctcaa ggatatcgcc agcgctaacg gcctggcctt
     2701 tcctaagatc accctgcctc cacagcctca caccaaagag ggcatcgagg cctacaacaa
     2761 cgtggtggcc cagatcgtga tttgggtcaa cctgaatctg tggcagaagc tgaagatcgg
     2821 cagggacgaa gccaagccac tgcagagact gaagggcttc cctagcttcc ctctggtgga
     2881 aagacaggcc aatgaagtgg attggtggga catggtctgc aacgtgaaga agctgatcaa
     2941 cgagaagaaa gaggatggca aggttttctg gcagaacctg gccggctaca agagacaaga
     3001 agccctgctg ccttacctga gcagcgaaga ggaccataaa aagcgggagg ggtctcttga
     3061 aaacccgaag aagttcgcca gataccagtt cggcgacctg ctgctgcacc tggaaaagaa
     3121 gcacggcgag gactggggca aagtgtacga tgaggcctgg gagagaatcg acaagaaggt
     3181 ggaaggcctg agcaagcaca ttaagctgga agaggaaaga aggagcgagg acgcccaatc
     3241 taaagccgct ctgaccgatt ggctgagagc caaggccagc tttgtgatcg agggcctgaa
     3301 agaggccgac aaggacgagt tctgcagatg cgagctgaag ctgcagaagt ggtacggcga
     3361 tctgagaggc aagcccttcg ccattgaggc cgagaacagc atcctggaca tcagcggctt
     3421 cagcaagcag tacaactgcg ccttcatttg gcagaaagac ggcgtcaaga aactgaacct
     3481 gtacctgatc atcaattact tcaaaggcgg caagctgcgg ttcaagaaga tcaaacccga
     3541 ggccttcgag gctaacagat tctacaccgt gatcaacaaa aagtccggcg agatcgtgcc
     3601 catggaagtg aacttcaact tcgacgaccc caacctgatt atcctgcctc tggccttcgg
     3661 caagagacag ggcagagagt tcatctggaa cgatctgctg agcctggaaa ccggctctct
     3721 gaagctggcc aatggcagag tgatcgagaa aaccctgtac aacaggagaa ccagacagga
     3781 cgagcctgct ctgtttgtgg ccctgacctt cgagagaaga gaggtgctgg acagcagcaa
     3841 catcaagccc atgaacctga tcggcatcga ccggggcgag aatatccctg ctgtgatcgc
     3901 cctgacagac cctgaaggat gcccactgag cagattcaag gactccctgg gcaaccctac
     3961 acacatcctg agaatcggcg agagctacaa agagaagcag aggacaatcc aggccgccaa
     4021 agaggtggaa cagagaagag ccggcggata ctctaggaag tacgccagca aggccaagaa
     4081 tctggccgac gacatggtcc gaaacaccgc cagagatctg ctgtactacg ccgtgacaca
     4141 ggacgccatg ctgatcttcg agaatctgag cagaggcttc ggccggcagg gcaagagaac
     4201 ctttatggcc gagaggcagt acaccagaat ggaagattgg ctcacagcta aactggccta
     4261 cgagggactg cccagcaaga cctacctgtc caaaacactg gcccagtata cctccaagac
     4321 ctgcagcaat tgcggcttca ccatcaccag cgccgactac gacagagtgc tggaaaagct
     4381 caagaaaacc gccaccggct ggatgaccac catcaacggc aaagagctga aggttgaggg
     4441 ccagatcacc tactacaaca ggtacaagag gcagaacgtc gtgaaggatc tgagcgtgga
     4501 actggacaga ctgagcgaag agagcgtgaa caacgacatc agcagctgga caaagggcag
     4561 atcaggcgag gctctgagcc tgctgaagaa gaggtttagc cacagacctg tgcaagagaa
     4621 gttcgtgtgc ctgaactgcg gcttcgagac acacgccgat gaacaggctg ccctgaacat
     4681 tgccagaagc tggctgttcc tgagaagcca agagtacaag aagtaccaga ccaacaagac
     4741 caccggcaac accgacaaga gggcctttgt ggaaacctgg cagagcttct acagaaaaaa
     4801 gctgaaagaa gtctggaagc ccgccgtgac tagtccaaaa aagaagagaa aggtagatta
     4861 caaagatgac gatgacaaag actacaagga tgatgatgat aagggatccg gcgccaccaa
     4921 cttcagcctg ctgaagcagg ccggcgacgt ggaggagaac cccggcccca tggtaagtaa
     4981 gggcgaagag gataatatgg cttctttgcc cgccacacac gaactgcaca tattcggatc
     5041 aataaacggg gtggatttcg atatggtcgg ccagggaaca ggaaacccta atgacggcta
     5101 cgaagagctc aatctcaaat ctaccaaagg ggacttacaa ttctctccat ggatcctagt
     5161 gccacatatt ggatatggct tccatcaata ccttccatat ccagacggta tgagcccctt
     5221 ccaggctgca atggtggacg ggtctggata tcaggttcac agaacaatgc aatttgagga
     5281 cggtgcatcc cttacagtta attacaggta cacatacgaa ggaagtcaca tcaaagggga
     5341 ggcgcaggtg aagggcaccg ggttcccagc tgatggacct gtaatgacaa attcactcac
     5401 agccgccgat tggtgcaggt caaagaaaac ctatcctaat gacaaaacaa ttatatccac
     5461 atttaaatgg tcttatacca caggaaatgg aaagagatac agatctacag ctagaacaac
     5521 ttacacattt gctaaaccaa tggcagccaa ctaccttaag aatcaaccaa tgtatgtgtt
     5581 cagaaagact gaactaaaac acagtaaaac agaacttaat ttcaaagagt ggcagaaggc
     5641 cttcacagac gttatgggta tggatgaact gtacaaggga tccggcgcaa caaacttctc
     5701 tctgctgaaa caagccggag atgtcgaaga gaatcctgga ccgaccgagt acaagcccac
     5761 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt
     5821 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
     5881 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     5941 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     6001 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     6061 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     6121 cgtcggagtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     6181 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa
     6241 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     6301 accgcgcacc tggtgcatga cccgcaagcc cggtgcctga acgcgttaag aattcctaga
     6361 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     6421 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     6481 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     6541 gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg gccgcaggaa
     6601 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     6661 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     6721 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     6781 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     6841 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     6901 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     6961 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     7021 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     7081 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     7141 accctatctc gggctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     7201 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     7261 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     7321 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     7381 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     7441 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     7501 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     7561 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     7621 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     7681 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     7741 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     7801 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     7861 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     7921 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     7981 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     8041 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     8101 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     8161 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     8221 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     8281 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     8341 ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag
     8401 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     8461 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     8521 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     8581 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     8641 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttc
//
