LOCUS       sequence-359734-        7889 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing mammalian codon optimized engineered DpbCasX-R3, 
            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 7889)
  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 7889)
  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: pCAT105
FEATURES             Location/Qualifiers
     source          1..7889
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      30..618
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     519..538
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        680..928
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     680..700
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     851..870
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        1062..1347
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     gap             1466..1481
                     /estimated_length=16
     intron          1593..1820
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1832..1841
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1844..1864
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4844..4864
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4865..4912
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             4922..4978
                     /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             4979..5575
                     /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     5472..5492
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5609..5626)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5615..5822
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5831..5971
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5831..5960
                     /label=AAV2 ITR
     rep_origin      6046..6501
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6133..6152)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6343..6364
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6518..6537)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6637..6659
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6697..6715)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6783..6887
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6888..6956,6957..7748)
                     /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(7106..7125)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
       61 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
      121 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
      181 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
      241 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
      301 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
      361 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
      421 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
      481 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
      541 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
      601 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
      661 tggccttttg ctcacatgtg agggcctatt tcccatgatt ccttcatatt tgcatatacg
      721 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
      781 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
      841 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
      901 atatatcttg tggaaaggac gaaacaccgg cgcttttatc tcattacttt gagagccatc
      961 accagcgact atgtcgtatg ggtaaagcgc ttatttatcg gagaaaccga taaataagaa
     1021 gcatcaaagg ggtcttcgag aagacccttt tttttggtac ccgttacata acttacggta
     1081 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat agtaacgcca
     1141 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     1201 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     1261 cccgcctggc attgtgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     1321 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
     1381 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
     1441 gcagcgatgg gggcgggggg gggggnnnnn nnnnnnnnnn naggcggaga ggtgcggcgg
     1501 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
     1561 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg
     1621 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc
     1681 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg agcaagaggt
     1741 aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg gagcacctgc
     1801 ctgaaatcac tttttttcag gttggaccgg tgccaccatg gccccaaaga agaagcggaa
     1861 ggtcagcgaa aagagaatca acaagatcag gaagaagctg agcgccgaca acgccaccaa
     1921 gcctgtgtct aggagtggcc ccatgaaaac cctgctcgtc agagtgatga ccgacgacct
     1981 gaagaagaga ctggaaaaga ggaggaagaa acccgaagtg atgccccaag tgatcagcaa
     2041 caacgccgcc aacaacctga gaatgctgct ggacgactac accaagatga aggaagccat
     2101 cctgcaggtc tactggcaag agttcaagga cgaccacgtg ggcctgatgt gcaagtttgc
     2161 ccagcctgcc agcaagaaga tcgaccagaa caagctgaag cccgagatgg acgagaaggg
     2221 caatctgacc acagccggct ttgcctgctc tcagtgtggc cagcctctgt tcgtgtacaa
     2281 gctggaacag gtgtccgaga aaggcaaggc ctacaccaac tacttcggca gatgtaacgt
     2341 ggccgagcac gagaagctga ttctgctggc ccagctgaaa cctgagaagg actctgatga
     2401 ggccgtgacc tacagcctgg gcaagtttgg acagagagcc ctggacttct acagcatcca
     2461 cgtgaccaaa gaaagcacac accccgtgaa gcccctggct cagatcgccg gcaatagata
     2521 cgcctctgga cctgtgggca aagccctgtc cgatgcctgc atgggaacaa tcgccagctt
     2581 cctgagcaag taccaggaca tcatcatcga gcaccagaag gtggtcaagg gcaaccagaa
     2641 gagactggaa agcctgaggg agctggccgg caaagagaac ctggaatacc ccagcgtgac
     2701 cctgcctcct cagcctcaca caaaagaagg cgtggacgcc tacaacgaag tgatcgccag
     2761 agtgagaatg tgggtcaacc tgaacctgtg gcagaagctg aaactgtcca gggacgacgc
     2821 caagcctctg ctgagactga agggcttccc tagcttcccc gtggtggaaa gaagggagaa
     2881 tgaggtggac tggtggaaca ccatcaacga agtcaagaaa ctgatcgatg ccaagagaga
     2941 catgggcaga gtgttttgga gcggagtgac cgccgagaag agaaacacca tcctggaagg
     3001 ctacaactac ctgcctaacg agaacgacca caagaagaga gagggcagcc tggaaaaccc
     3061 taagaagccc gccaagagac agttcggcga cctgctgctg tacctggaga agaagtatgc
     3121 cggcgactgg ggcaaagtgt tcgatgaggc ctgggagaga atcgacaaga agattgccgg
     3181 cctgaccagc cacatcgaga gagaggaagc cagaaacgcc gaggacgccc agtctaaagc
     3241 cgtgctgaca gattggctga gagccaaggc cagcttcgtg ctggaaaggc tgaaagaaat
     3301 ggatgagaaa gagttctacg cctgcgagat ccagctgcag aagtggtacg gggacctgag
     3361 aggcaatccc tttgccgtgg aagccgagaa cagagtggtg gacatcagcg gctttagcat
     3421 cggctctgac ggccactcca tccagtacag gaacctgctg gcctggaagt acctggaaaa
     3481 tggcaagaga gagttctacc tgctgatgaa ctacggcaag aagggcagaa tcagattcac
     3541 cgacggcacc gacatcaaga aaagcggcaa gtggcagggc ctgctgtatg gcggcggaaa
     3601 ggccaaagtg atcgacctga ccttcgatcc cgacgacgag cagctgatca ttctgcctct
     3661 ggccttcggc accagacagg gcagagagtt catctggaac gatctgctgt ccctggaaac
     3721 cggcctgatc aagctggcca acggcagggt gatcgagaaa accatctaca acaagaaaat
     3781 cggcagagat gagcccgcac tgtttgtggc cctgaccttt gagaggagag aagtggtcga
     3841 tcccagcaac atcaagcccg tgaacctgat cggagtggac agaggcgaga atatccctgc
     3901 cgtgattgcc ctgacagacc ctgagggatg tcccctgcct gagtttaagg atagctctgg
     3961 cggccctacc gacatcctga gaatcggcga gggctacaaa gagaagcaga gagccatcca
     4021 ggccgccaaa gaggtggaac agagaagagc cggcggatac agcagaaagt tcgccagcaa
     4081 gtccagaaac ctggccgatg acatggtcag gaacagcgcc agggacctgt tctaccacgc
     4141 cgtgacacat gatgccgtgc tggtgttcga gaacctgtca agaggcttcg gcagacaggg
     4201 aaaaagaacc ttcatgaccg agagacagta cacgaagatg gaagattggc tcaccgccaa
     4261 actggcctac gagggcctga caagcaagac ctacctgtcc aagacactgg cccagtacac
     4321 ctccaagacc tgctccaatt gcggcttcac catcaccacc gccgactacg atggaatgct
     4381 cgtgagactg aagaaaacct ccgacggctg ggccaccaca ctgaacaaca aagagctgaa
     4441 agccgagggc cagatcacct actacaacag atacaagagg cagaccgtcg agaaagagct
     4501 gtccgccgaa ctcgacagac tgagcgagga atccggcaac aacgacatca gcaagtggac
     4561 caagggacgc agagatgagg ccctgttcct gctgaagaag aggttcagcc acagacctgt
     4621 gcaagagcag tttgtgtgcc tggattgcgg ccatgaagtg cacgcagatg aacaggccgc
     4681 tctgaatatc gccagaagct ggctgttcct gcgttctcag gagtataaga agtatcagac
     4741 gaataagacc acagggaaca cggataaacg ggccttcgtc gaaacatggc agtccttcta
     4801 caagagaagg ctcaaagaag tgtggaagcc caacgccact agtccaaaaa agaagagaaa
     4861 ggtagattac aaagatgacg atgacaaaga ctacaaggat gatgatgata agggatccgg
     4921 cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgac
     4981 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     5041 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     5101 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     5161 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     5221 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     5281 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     5341 gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     5401 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     5461 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     5521 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg
     5581 ttaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     5641 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     5701 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     5761 ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg
     5821 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     5881 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     5941 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     6001 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     6061 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     6121 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     6181 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     6241 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     6301 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     6361 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     6421 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     6481 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     6541 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     6601 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     6661 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     6721 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     6781 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     6841 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     6901 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     6961 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     7021 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     7081 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     7141 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     7201 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     7261 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     7321 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     7381 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     7441 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     7501 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     7561 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg
     7621 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     7681 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     7741 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     7801 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     7861 aacgtgagtt ttcgttccac tgagcgtca
//
